scholarly journals Computational design of Small Transcription Activating RNAs (STARs) for versatile and dynamic gene regulation

2017 ◽  
Author(s):  
James Chappell ◽  
Alexandra Westbrook ◽  
Matthew Verosloff ◽  
Julius B. Lucks
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
Gene Activation ◽  
Computational Design ◽  
Cellular Functions ◽  
Transcription Control ◽  
Control Of Gene Expression ◽  
High Performing ◽  
The Creation ◽  
Cellular Phenotypes

AbstractA longstanding goal of synthetic biology has been the programmable control of cellular functions. Central to this goal is the creation of versatile regulatory toolsets that allow for programmable control of gene expression. Of the many regulatory molecules available, RNA regulators offer the intriguing possibility of de novo design – allowing for the bottom-up molecular-level design of genetic control systems. Here we present a computational design approach for the creation of a bacterial regulator called Small Transcription Activating RNAs (STARs) and create a library of high-performing and orthogonal STARs that achieve up to ∼9000-fold gene activation. We then demonstrate the versatility of RNA-based transcription control by showing the broad utility of STARs – from acting synergistically with existing constitutive and inducible regulators, to reprogramming cellular phenotypes and controlling multigene metabolic pathway expression. Finally, we combine these new STARs with themselves and CRISPRi transcriptional repressors to deliver new types of RNA-based genetic circuitry that allow for sophisticated and temporal control of gene expression.

scholarly journals Computational design and experimental characterization of a photo-controlled mRNA-cap guanine-N7 methyltransferase

2021 ◽  
Author(s):  
Dennis Reichert ◽  
Helena Schepers ◽  
Julian Simke ◽  
Horst Lechner ◽  
Wolfgang Dörner ◽  
...  
Keyword(s):  
Gene Expression ◽  
Computational Design ◽  
Eukaryotic Cells ◽  
Transcriptional Level ◽  
Experimental Characterization ◽  
Temporal Control ◽  
Control Of Gene Expression ◽  
Multicellular Organisms

The spatial and temporal control of gene expression at the post-transcriptional level is essential in eukaryotic cells and developing multicellular organisms. In recent years optochemical and optogenetic tools have enabled...

scholarly journals De novo design of programmable inducible promoters

2019 ◽  
Vol 47 (19) ◽  
pp. 10452-10463 ◽  
Author(s):  
Xiangyang Liu ◽  
Sanjan T P Gupta ◽  
Devesh Bhimsaria ◽  
Jennifer L Reed ◽  
José A Rodríguez-Martínez ◽  
...  
Keyword(s):  
Gene Expression ◽  
High Throughput Screening ◽  
De Novo ◽  
Vital Role ◽  
Control Of Gene Expression ◽  
Core Motif ◽  
Expression Levels ◽  
Synthetic Promoters ◽  
The Core ◽  
Rich Diversity

Abstract Ligand-responsive allosteric transcription factors (aTF) play a vital role in genetic circuits and high-throughput screening because they transduce biochemical signals into gene expression changes. Programmable control of gene expression from aTF-regulated promoter is important because different downstream effector genes function optimally at different expression levels. However, tuning gene expression of native promoters is difficult due to complex layers of homeostatic regulation encoded within them. We engineered synthetic promoters de novo by embedding operator sites with varying affinities and radically reshaped binding preferences within a minimal, constitutive Escherichia coli promoter. Multiplexed cell-based screening of promoters for three TetR-like aTFs generated with this approach gave rich diversity of gene expression levels, dynamic ranges and ligand sensitivities and were 50- to 100-fold more active over their respective native promoters. Machine learning on our dataset revealed that relative position of the core motif and bases flanking the core motif play an important role in modulating induction response. Our generalized approach yields customizable and programmable aTF-regulated promoters for engineering cellular pathways and enables the discovery of new small molecule biosensors.

scholarly journals A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana.

2021 ◽  
Author(s):  
Elena Garcia-Perez ◽  
Borja Diego-Martin ◽  
Alfredo Quijano-Rubio ◽  
Elena Moreno Gimenez ◽  
Diego Orzaez ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcriptional Activation ◽  
Nicotiana Benthamiana ◽  
Gene Networks ◽  
Rna Binding ◽  
Gene Activation ◽  
Dependent Manner ◽  
Copper Binding ◽  
Control Of Gene Expression ◽  
Guide Rna

CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS). In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations in place, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold for the endogenous N. benthamiana DFR gene, with negligible expression in the absence of the trigger. The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.

scholarly journals SUMO Paralog Specific Functions Revealed through Systematic Analysis of Human Knockout Cell Lines and Gene Expression Data

2021 ◽  
pp. mbc.E21-01-0031
Author(s):  
Danielle Bouchard ◽  
Wei Wang ◽  
Wei-Chih Yang ◽  
Shuying He ◽  
Anthony Garcia ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cell Lines ◽  
Genotoxic Stress ◽  
Nuclear Body ◽  
Human Tissues ◽  
Cellular Functions ◽  
Systematic Analysis ◽  
Control Of Gene Expression ◽  
Knock Out ◽  
Sequence Homologies

The small ubiquitin-related modifiers (SUMOs) regulate nearly every aspect of cellular function, from gene expression in the nucleus to ion transport at the plasma membrane. In humans, the SUMO pathway has five SUMO paralogs with sequence homologies that range from 45% to 97%. SUMO1 and SUMO2 are the most distantly related paralogs, and also the best studied. To what extent SUMO1, SUMO2 and the other paralogs impart unique and non-redundant effects on cellular functions, however, has not been systematically examined and is therefore not fully understood. For instance, knockout studies in mice have revealed conflicting requirements for the paralogs during development and studies in cell culture have relied largely on transient paralog overexpression or knockdown. To address the existing gap in understanding, we first analyzed SUMO paralog gene expression levels in normal human tissues and found unique patterns of SUMO1-3 expression across 30 tissue types, suggesting paralog-specific functions in adult human tissues. To systematically identify and characterize unique and non-redundant functions of the SUMO paralogs in human cells, we next used CRISPR-Cas9 to knock out SUMO1 and SUMO2 expression in osteosarcoma (U2OS) cells. Analysis of these knockout cell lines revealed essential functions for SUMO1 and SUMO2 in regulating cellular morphology, PML nuclear body structure, responses to proteotoxic and genotoxic stress, and control of gene expression. Collectively, our findings reveal non-redundant regulatory roles for SUMO1 and SUMO2 in controlling essential cellular processes and provide a basis for more precise SUMO-targeting therapies.

scholarly journals A Synthetic Transcription Platform for Programmable Gene Expression in Mammalian Cells

2020 ◽  
Author(s):  
William C.W. Chen ◽  
Leonid Gaidukov ◽  
Ming-Ru Wu ◽  
Jicong Cao ◽  
Gigi C.G. Choi ◽  
...  
Keyword(s):  
Gene Expression ◽  
Interferon Gamma ◽  
Mammalian Cells ◽  
Cell Types ◽  
Regulatory Elements ◽  
Genetic Circuits ◽  
Control Of Gene Expression ◽  
Transcription System ◽  
Multiple Cell ◽  
Cellular Phenotypes

Precise, scalable, and sustainable control of genetic and cellular activities in mammalian cells is key to developing precision therapeutics and smart biomanufacturing. We created a highly tunable, modular, versatile CRISPR-based synthetic transcription system for the programmable control of gene expression and cellular phenotypes in mammalian cells. Genetic circuits consisting of well-characterized libraries of guide RNAs, binding motifs of synthetic operators, transcriptional activators, and additional genetic regulatory elements expressed mammalian genes in a highly predictable and tunable manner. We demonstrated the programmable control of reporter genes episomally and chromosomally, with up to 25-fold more EF1[alpha]; promoter activity, in multiple cell types. We used these circuits to program secretion of human monoclonal antibodies and to control T cell effector function marked by interferon-[gamma] production. Antibody titers and interferon-[gamma]; concentrations were significantly correlated with synthetic promoter strengths, providing a platform for programming gene expression and cellular function for biological, biomanufacturing, and biomedical applications.

scholarly journals Characterization of five purine riboswitches in cellular and cell-free expression systems

2021 ◽  
Author(s):  
Milca Rachel da Costa Ribeiro Lins ◽  
Graciely Gomes Correa ◽  
Laura Araujo da Silva Amorim ◽  
Rafael Augusto Lopes Franco ◽  
Nathan Vinicius Ribeiro ◽  
...  
Keyword(s):  
Gene Expression ◽  
De Novo ◽  
In Vitro Transcription ◽  
Vitro Assay ◽  
Control Of Gene Expression ◽  
Transcription Terminator ◽  
Variable Expression ◽  
Expression Platform

Bacillus subtilis employs five purine riboswitches for the control of purine de novo synthesis and transport at the transcription level. All of them are formed by a structurally conserved aptamer, and a variable expression platform harboring a rho-independent transcription terminator. In this study, we characterized all five purine riboswitches under the context of active gene expression processes both in vitro and in vivo. We identified transcription pause sites located in the expression platform upstream of the terminator of each riboswitch. Moreover, we defined a correlation between in vitro transcription readthrough and in vivo gene expression. Our in vitro assay demonstrated that the riboswitches operate in the micromolar range of concentration for the cognate metabolite. Our in vivo assay showed the dynamics of control of gene expression by each riboswitch. This study deepens the knowledge of the regulatory mechanism of purine riboswitches.

scholarly journals Differential nuclear import sets the timing of protein access to the embryonic genome

2021 ◽  
Author(s):  
Thao Nguyen ◽  
Eli Costa ◽  
Tim Deibert ◽  
Jose Reyes ◽  
Felix Keber ◽  
...  
Keyword(s):  
Gene Expression ◽  
Early Development ◽  
Control Cell ◽  
Gene Activation ◽  
Nuclear Proteins ◽  
Control Of Gene Expression ◽  
Embryonic Genome ◽  
Regulatory Cascades ◽  
Protein Entry ◽  
Nuclear Proteome

The development of a fertilized egg to an embryo requires the proper temporal control of gene expression. During cell differentiation, timing is often controlled via cascades of transcription factors (TFs). However, in early development, transcription is often inactive, and many TF levels are constant, suggesting that unknown mechanisms govern the observed rapid and ordered onset of gene expression. Here, we find that in early embryonic development, access of maternally deposited nuclear proteins to the genome is temporally ordered via importin affinities, thereby timing the expression of downstream targets. We quantify changes in the nuclear proteome during early development and find that nuclear proteins, such as TFs and RNA polymerases, enter nuclei sequentially. Moreover, we find that the timing of the access of nuclear proteins to the genome corresponds to the timing of downstream gene activation. We show that the affinity of proteins to importin is a major determinant in the timing of protein entry into embryonic nuclei. Thus, we propose a mechanism by which embryos encode the timing of gene expression in early development via biochemical affinities. This process could be critical for embryos to organize themselves before deploying the regulatory cascades that control cell identities.

scholarly journals Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction

2021 ◽  
Author(s):  
Emilia Dimitrova ◽  
Angelika Feldmann ◽  
Robin H van der Weide ◽  
Koen D Flach ◽  
Anna Lastuvkova ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Activation ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Gene Promoters ◽  
Control Of Gene Expression ◽  
Precise Control ◽  
3D Genome ◽  
Gene Regulatory Elements ◽  
Gene Regulatory

Precise control of gene expression underpins normal development. This relies on mechanisms that enable communication between gene promoters and other regulatory elements. In embryonic stem cells (ESCs), the CDK-Mediator (CDK-MED) complex has been reported to physically link gene regulatory elements to enable gene expression and also prime genes for induction during differentiation. Here we discover that CDK-MED contributes little to 3D genome organisation in ESCs, but has a specific and essential role in controlling interactions between inactive gene regulatory elements bound by Polycomb repressive complexes (PRCs). These interactions are established by the canonical PRC1 (cPRC1) complex but rely on CDK-MED, which facilitates binding of cPRC1 to its target sites. Importantly, through separation of function experiments, we reveal that this collaboration between CDK-MED and cPRC1 in creating long-range interactions does not function to prime genes for induction during differentiation. Instead, we discover that priming relies on an interaction-independent mechanism whereby the CDK module supports core Mediator engagement with gene promoters to enable gene activation.

A second paradigm for gene activation in bacteria

2006 ◽  
Vol 34 (6) ◽  
pp. 1067-1071 ◽  
Author(s):  
M. Buck ◽  
D. Bose ◽  
P. Burrows ◽  
W. Cannon ◽  
N. Joly ◽  
...  
Keyword(s):  
Gene Expression ◽  
Conformational Changes ◽  
Atp Hydrolysis ◽  
Gene Activation ◽  
Control Of Gene Expression ◽  
Functional Studies ◽  
Promoter Complex ◽  
Molecular Machinery ◽  
Dna Template ◽  
Rna Polymerase Holoenzyme

Control of gene expression is key to development and adaptation. Using purified transcription components from bacteria, we employ structural and functional studies in an integrative manner to elaborate a detailed description of an obligatory step, the accessing of the DNA template, in gene expression. Our work focuses on a specialized molecular machinery that utilizes ATP hydrolysis to initiate DNA opening and permits a description of how the events triggered by ATP hydrolysis within a transcriptional activator can lead to DNA opening and transcription. The bacterial EBPs (enhancer binding proteins) that belong to the AAA+ (ATPases associated with various cellular activities) protein family remodel the RNAP (RNA polymerase) holoenzyme containing the σ54 factor and convert the initial, transcriptionally silent promoter complex into a transcriptionally proficient open complex using transactions that reflect the use of ATP hydrolysis to establish different functional states of the EBP. A molecular switch within the model EBP we study [called PspF (phage shock protein F)] is evident, and functions to control the exposure of a solvent-accessible flexible loop that engages directly with the initial RNAP promoter complex. The σ54 factor then controls the conformational changes in the RNAP required to form the open promoter complex.

scholarly journals De novo Design of Translational RNA Repressors

2018 ◽  
Author(s):  
Paul D. Carlson ◽  
Cameron J. Glasscock ◽  
Julius B. Lucks
Keyword(s):  
Gene Expression ◽  
Synthetic Biology ◽  
De Novo ◽  
Genetic Circuit ◽  
Bacterial Cells ◽  
Control Of Gene Expression ◽  
Sequence Characterization ◽  
Mrna Targets ◽  
Modern Drug ◽  
And Function

ABSTRACTA central goal of synthetic biology is the development of methods for the predictable control of gene expression. RNA is an attractive substrate by which to achieve this goal because the relationship between its sequence, structure, and function is being uncovered with increasing depth. In addition, design approaches that use this relationship are becoming increasingly effective, as evidenced by significant progress in the de novo design of RNA-based gene regulatory mechanisms that activate transcription and translation in bacterial cells. However, the design of synthetic RNA mechanisms that are efficient and versatile repressors of gene expression has lagged, despite their importance for gene regulation and genetic circuit construction. We address this gap by developing two new classes of RNA regulators, toehold repressors and looped antisense oligonucleotides (LASOs), that repress translation of a downstream gene in response to an arbitrary input RNA sequence. Characterization studies show that these designed RNAs robustly repress translation, are highly orthogonal, and can be multiplexed with translational activators. We show that our LASO design can repress endogenous mRNA targets and distinguish between closely-related genes with a high degree of specificity and predictability. These results demonstrate significant yet easy-to-implement improvements in the design of synthetic RNA repressors for synthetic biology, and point more broadly to design principles for repressive RNA interactions relevant to modern drug design.

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